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Biomarkers for early diagnosis of pancreatic cancer are greatly needed, as the high fatality of this cancer is in part due to delayed detection. α1,4-linked N-acetylglucosamine (αGlcNAc), a unique O-glycan specific to gastric gland mucus, is biosynthesized by α1,4-N-acetylglucosaminyltransferase (α4GnT) and primarily bound at the terminal glycosylated residue to scaffold protein MUC6. We previously reported that αGlcNAc expression decreases at early stages of neoplastic pancreatic lesions, followed by decreased MUC6 expression, although functional effects of these outcomes were unknown. Here, we ectopically expressed α4GnT, the αGlcNAc biosynthetic enzyme, together with MUC6 in the human pancreatic cancer cell lines MIA PaCa-2 and PANC-1, neither of which expresses α4GnT and MUC6. We observed significantly suppressed proliferation in both lines following coexpression of α4GnT and MUC6. Moreover, cellular motility decreased following MUC6 ectopic expression, an effect enhanced by cotransduction with α4GnT. MUC6 expression also attenuated invasiveness of both lines relative to controls, and this effect was also enhanced by additional α4GnT expression. We found αGlcNAc-bound MUC6 formed a complex with trefoil factor 2. Furthermore, analysis of survival curves of patients with pancreatic ductal adenocarcinoma using a gene expression database showed that samples marked by higher A4GNT or MUC6 mRNA levels were associated with relatively favorable prognosis. These results strongly suggest that αGlcNAc and MUC6 function as tumor suppressors in pancreatic cancer and that decreased expression of both may serve as a biomarker of tumor progression to pancreatic cancer.

Gastric gland mucin consists of core protein MUC6 with residues heavily glycosylated by unique O-glycans carrying α1,4-linked N-acetylglucosamine (αGlcNAc). αGlcNAc-glycosylated MUC6 protein is seen in normal gastric and duodenal glands. Decreased αGlcNAc glycosylation on MUC6-positive tumor cells is often observed in premalignant lesions of the stomach, pancreas, and bile duct, and decreased MUC6 expression is seen in invasive cancer of these organs. Lung cancer (LC) is the most common cause of cancer death worldwide. Recently, the adenocarcinoma subtype has become the most common histological subtype of LC, and one of its invasive forms is invasive mucinous adenocarcinoma (IMA). Currently, prognostic markers of LC IMA are unknown. Here, we analyzed MUC5AC, MUC6, and αGlcNAc expression in 54 IMA LC cases. MUC5AC was positively expressed in 50 (93%), MUC6 in 38 (70%), and αGlcNAc in 19 (35%). Each expression level was scored from 0 to 3. The αGlcNAc expression score was significantly decreased relative to MUC6 (P 0.001). Interestingly, disease-free survival was significantly higher in MUC6-positive than MUC6-negative cases based on the log-rank test (P = 0.021). For in vitro analysis, we ectopically expressed MUC6 in A549 cells, derived from LC and harboring a KRAS mutation. MUC6-expressing A549 cells showed significantly lower proliferation, motility, and invasiveness than control cells. Finally, F-actin staining in MUC6-expressing cells revealed a decrease or loss of filopodia associated with decreased levels of FSCN transcripts, which encodes an actin-bundling protein fascin1 necessary for cell migration. We conclude that MUC6 expression is a preferable prognostic biomarker in IMA LC.

Biliary tract cancer (BTC) is typically lethal due to the difficulty of early stage diagnosis. Thus, novel biomarkers of BTC precursors are necessary. Biliary intraepithelial neoplasia (BilIN) is a major precursor of BTC and is classified as low or high grade based on cell atypia. In normal gastric mucosa, gastric gland mucin‐specific O‐glycans are unique in having α1,4‐linked N‐acetylglucosamine (αGlcNAc) attached to MUC6. Previously, we reported that αGlcNAc functions as a tumor suppressor of differentiated‐type gastric adenocarcinoma and that decreased αGlcNAc glycosylation on MUC6 in gastric, pancreatic, and uterine cervical neoplasms occurs in cancer as well as in their precursor lesions. However, αGlcNAc and MUC6 expression patterns in biliary tract neoplasms have remained unclear. Here, we analyzed MUC5AC, MUC6, and αGlcNAc expression status in 51 BTC cases and compared the expression of each with progression from low‐grade BilIN to invasive adenocarcinoma (IAC). The frequency of αGlcNAc‐positive and MUC6‐positive lesions decreased with tumor progression. When we compared each marker’s expression level with tumor progression, we found that the MUC6 expression score in IAC was significantly lower than in low‐grade or high‐grade BilIN (P, Alpha‐1,4‐linked N‐acetylglucosamine glycosylation on MUC6 is already decreased in biliary intraepithelial neoplasia.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/ Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/ Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/ Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/ Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/ Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/ Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development

Objectives To evaluate the expression of annexin A1 protein in patients with renal cell carcinoma. Methods Annexin A1 expression was examined in renal cell carcinoma specimens from 27 patients, and their disease-free survival was analyzed using the log-rank test. Annexin A1 knockdown in the human renal cell carcinoma cell line Caki-1 was carried out, and its proliferation, invasion, motility and adhesion were compared with those of control cells. Results In 13 out of 27 patients, annexin A1 was highly expressed in the membrane of renal cell carcinoma tumor cells, whereas in the rest of the patients, annexin A1 expression was weak or negligible in the membrane of those cells. Patients with high annexin A1 expression had significantly poorer disease-free survival than those with weak or negligible annexin A1 expression (P = 0.031). In the renal cell carcinoma cell line, annexin A1 knockdown cells showed significantly decreased proliferation, invasion, motility and adhesion relative to control cells, and expressed lower relative levels of membrane-type 1 matrix metalloproteinase and hypoxia-inducible factor 1-alpha transcripts, showing a potential pathway regulated by annexin A1. Conclusion Annexin A1 is associated with renal cell carcinoma malignant potential and could serve as a marker of poor prognosis.

Aims Gastric neoplasms showing oxyntic gland differentiation (GAOGs) constitute a gastric neoplasm subtype that shows low atypia, thus similar to non-neoplastic gastric oxyntic glands. Therefore, their diagnosis in biopsy specimens is difficult. GAOGs were first described in 2007, and introduced in the latest World Health Organization classification book as gastric adenocarcinoma of the fundic gland type (GA-FG) and oxyntic gland adenoma. Previously, we assessed α1,4-linked N-acetylglucosamine (αGlcNAc) residues attached to the MUC6 scaffold in gastric neoplasms, and observed decreased αGlcNAc glycosylation in both differentiated-type gastric cancer and high-grade pyloric gland adenoma (PGA), a gastric cancer precursor. GA-FG and PGA often harbour the same mutations. However, the αGlcNAc status in GAOGs remained unknown. To elucidate αGlcNAc expression in GAOGs, we performed the study. Methods and results We assessed the expression of αGlcNAc; the mucin markers MUC6, MUC5AC, and MUC2; the gastric gland cell markers MIST1, pepsinogen 1 (PG1), H/K-ATPase and chromogranin-A (CGA); and the proliferation marker Ki67 in 13 GAOG lesions. All 13 (100%) were MUC6-positive, whereas 10 (76.2%) were αGlcNAc-negative. Moreover, all 13 (100%) were MIST1- and PG1-positive, three (23.1%) were MUC5AC-positive, four (30.8%) were H/K-ATPase-positive, and one (7.7%) was CGA-positive. Conclusions GAOGs frequently lost αGlcNAc residues on MUC6, but expressed the gastric gland progenitor marker MIST1 and aberrantly expressed various types of gastric gland cell lineage marker, suggestive of immature differentiation to gastric gland cells. Thus, diffuse MIST1 positivity and decreased αGlcNAc glycosylation on MUC6-positive cells could serve as important biomarkers for the histopathological diagnosis of GAOG.

LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.

Gastric gland mucin-specific O-glycans are unique in having α1,4-linked N-acetylglucosamine (αGlcNAc) attached to MUC6. We previously reported decreased expression of αGlcNAc relative to MUC6 in gastric and pancreatic neoplasms, but its significance in cervical glandular lesions remained unclear. Here, we analyzed MUC5AC, MUC6, αGlcNAc, and p16 expression in 9 lesions of mucinous carcinoma, gastric type with minimal deviation adenocarcinoma (GAS-MDA), 5 of GAS with nonMDA (GAS-nonMDA), 14 of typical lobular endocervical gland hyperplasia (LEGH), and 5 of atypical LEGH (33 total lesions). All 33 were MUC5AC-positive. Moreover, all 14 typical LEGH, 5 atypical LEGH, 8 of 9 GAS-MDA, and 3 of 5 GAS-nonMDA were MUC6-positive. All 14 typical LEGH, 2 of 5 atypical LEGH, 3 of 9 GAS-MDA, and 1 of 5 GAS-nonMDA were αGlcNAc-positive. The proportion of αGlcNAc-positive atypical LEGH or GAS-MDA or GAS-nonMDA lesions was significantly smaller than that seen in typical LEGH lesions (P

Pancreatic cancer is lethal, as it is often detected late. Thus novel biomarkers of precursor lesions are needed to devise timely therapies. Pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) are major precursors of pancreatic cancer. In normal gastric mucosa, gastric gland mucin-specific O-glycans are unique in having α1,4-linked N-acetylglucosamine (αGlcNAc) residues attached to MUC6. Recently we reported that αGlcNAc functions as a tumor suppressor for differentiated-type gastric adenocarcinoma (Karasawa et al. J Clin Invest 122, 923-934, 2012). MUC6 is also expressed in pancreatic neoplasms including PanIN and IPMN, but the role of αGlcNAc expression in pancreatic neoplasms remains unknown. Here, we analyze expression patterns of αGlcNAc, MUC6 and MUC5AC in pancreatic neoplasms and compare them with progression from PanIN to invasive ductal adenocarcinoma (IDAC) (the PanIN-IDAC sequence; 20 cases) and from IPMN to IPMN with associated invasive carcinoma (IPMNAIC) (the IPMN-IPMNAIC sequence; 20 cases). At both sequences, the frequency of MUC6-positive and αGlcNAc-positive lesions decreased with tumor progression. We then compared expression levels of αGlcNAc and MUC6 at each step of the progression. At the PanIN-IDAC sequence, αGlcNAc expression significantly decreased relative to MUC6 in low-grade PanIN (P = 0.021), high-grade PanIN/intraductal spread of IDAC (P = 0.031), and IDAC (P = 0.013). At the IPMN-IPMNAIC sequence, decreased αGlcNAc expression was also observed in low-grade IPMN exhibiting gastric-type morphology (P = 0.020). These results suggest that decreased expression of αGlcNAc relative to MUC6 occurs early and marks the initiation of tumor progression to pancreatic cancer. This article is protected by copyright. All rights reserved.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development

Gastric gland mucin secreted from pyloric gland cells, mucous neck cells, and cardiac gland cells of the gastric mucosa harbors unique O-glycans carrying terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc), which are primarily attached to the scaffold mucin core protein MUC6. αGlcNAc acts as an antibiotic against Helicobacter pylori (H. pylori), a microbe causing gastric cancer. In addition, mice deficient in A4gnt, which encodes the enzyme α1,4-N-acetylglucosaminyltransferase (α4GnT) that catalyzes αGlcNAc biosynthesis, spontaneously develop gastric differentiated-type adenocarcinoma, even if not infected by H. pylori. Thus, αGlcNAc prevents gastric cancer as both an antibiotic and a tumor suppressor (Nakayama in Acta Histochem Cytochem 47:1-9, 2014b). Indeed, in humans αGlcNAc loss on MUC6 in differentiated-type adenocarcinoma is closely associated with poor patient prognosis (Shiratsu et al. in Cancer Sci 105:126-133, 2014). Recently, we reported reduced αGlcNAc expression on MUC6 in both pyloric gland adenoma of the stomach and chronic atrophic gastritis, in Barrett's esophagus, and in pancreatic intraductal papillary-mucinous neoplasm (IPMN)/pancreatic intraepithelial neoplasia (PanIN), all potentially premalignant conditions. This review discusses whether relatively reduced levels of αGlcNAc in these lesions could serve as a biomarker to predict malignant potential and cancer progression.

LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.

Summary Single-CpG resolution genome-wide DNA methylation analysis indicated that distinct DNA methylation profiles are established during field cancerization in gastric mucosae, and such profiles at the precancerous stage are inherited by gastric cancers, thus determining tumor aggressiveness and patient outcome., The aim of this study was to clarify the significance of DNA methylation alterations during gastric carcinogenesis. Single-CpG resolution genome-wide DNA methylation analysis using the Infinium assay was performed on 109 samples of non-cancerous gastric mucosa (N) and 105 samples of tumorous tissue (T). DNA methylation alterations in T samples relative to N samples were evident for 3861 probes. Since N can be at the precancerous stage according to the field cancerization concept, unsupervised hierarchical clustering based on DNA methylation levels was performed on N samples (βN) using the 3861 probes. This divided the 109 patients into three clusters: A (n = 20), B1 (n = 20), and B2 (n = 69). Gastric carcinomas belonging to Cluster B1 showed tumor aggressiveness more frequently than those belonging to Clusters A and B2. The recurrence-free and overall survival rates of patients in Cluster B1 were lower than those of patients in Clusters A and B2. Sixty hallmark genes for which βN characterized the epigenetic clustering were identified. We then focused on DNA methylation levels in T samples (βT) of the 60 hallmark genes. In 48 of them, including the ADAM23, OLFM4, AMER2, GPSM1, CCL28, DTX1 and COL23A1 genes, βT was again significantly correlated with tumor aggressiveness, and the recurrence-free and/or overall survival rates. Multivariate analyses revealed that βT was a significant prognostic factor, being independent of clinicopathological parameters. These data indicate that DNA methylation profiles at the precancerous stage may be inherited by gastric carcinomas themselves, thus determining tumor aggressiveness and patient outcome.

Gastric cancer is one of the most common malignancies worldwide and patients with advanced gastric cancer still have poor clinical outcomes. The overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) mRNA in colorectal cancer and its correlation with clinicopathological factors were recently reported by us. In this study, we show LGR5 mRNA overexpression in human gastric cancer specimens by quantitative RT-PCR and in situ hybridization and assess a correlation with clinicopathological factors. The mean expression of LGR5 mRNA in cancerous tissues was five times higher than that in normal tissue (P = 0.0002). Furthermore, LGR5 mRNA expression show marked variation among cases and significantly increased in cases where lymphatic invasion was present compared with those where it was absent (P = 0.0056). Although the mean expression level of LGR5 was observed to be higher in nodal metastasis and venous invasion positive cases compared to negative cases, a significant difference was not observed. These results suggest that LGR5 can be a biomarker for malignancy in gastric cancer.

Few cases of gastric carcinoma with infiltrating multi-nucleated giant cells (MGCs) have been reported, and giant cells infiltrating the gastric carcinoma were previously described as osteoclast-like giant cells (OGCs). However, hepatoid adenocarcinomas have never been reported previously, and the present case is extremely rare. A 100-year-old Japanese man with gastralgia was found to have a mass in his gastric body. Histological examination showed a poorly differentiated adenocarcinoma with infiltration of MGCs. Vascular and lymphatic invasion were noted but there were no metastases. Almost all the tumor comprised cells with hepatoid-like features. The MGCs proliferated, with infiltration of lymphoid cells. A few MGCs contained mucous material in their cytoplasm, indicating these were foreign-body giant cells. Immunohistochemically, the hepatoid-like components were positive for AFP, and staining with polyclonal antibodies against carcinoembryonic antigen (CEA) showed the canalicular pattern. We concluded that this component was hepatoid adenocarcinoma. The MGCs were positive for CD68, and surrounding infiltrating lymphoid cells were diffusely positive for CD3. Infiltration of MGCs in gastric cancer may represent a cellular immune response against gastric cancer invasion. Further studies are required to elucidate the etiology of MGCs in gastric cancer.

Aims Pyloric gland adenoma (PGA) is a unique gastric neoplasm expressing mucin 6 (MUC6), and is often associated with high-grade dysplasia and/or adenocarcinoma. MUC6 secreted from the gastric gland mucous cells, such as pyloric gland cells, carries unique O-glycans with terminal α1,4-linked N-acetylglucosamine (αGlcNAc) residues on its molecule. As we recently demonstrated that αGlcNAc serves as a tumour suppressor for gastric adenocarcinoma, this study aimed to investigate the significance of αGlcNAc expression in PGA. Methods and results Eighteen patients with PGA were examined with immunohistochemistry for αGlcNAc and MUC6. αGlcNAc and MUC6 were coexpressed in 12 of 18 PGAs. However, reduced αGlcNAc expression relative to MUC6 expression was observed in six cases. When the MIB-1 labelling index (LI) of tumour cells was examined with respect to reduced αGlcNAc expression, the MIB-1 LI was significantly higher in PGAs showing decreased αGlcNAc expression relative to MUC6 expression than in PGAs with unchanged αGlcNAc expression (P = 0.023). Conclusions The present study indicates that coexpression of αGlcNAc and MUC6 in PGA suggests the presence of fully glycosylated MUC6 on tumour cells, consistent with pyloric gland differentiation. However, the decreased glycosylation of αGlcNAc on MUC6 is associated with high mitotic activity of tumour cells, indicative of malignant potential of PGA.

Multiple gastrointestinal stromal tumors (GISTs) caused by germline c-kit gene mutations are an extremely rare autosomal dominant disorder. A 57-year-old Japanese woman was referred to a hospital for appetite loss and severe weight loss. She had 2 large abdominal masses around the stomach, which were surgically resected. Histological examination revealed that these tumors were GISTs. Multiple microscopic GISTs and diffuse hyperplasia of the interstitial cells of Cajal were also seen in the background gastric and small intestinal walls. Characteristically, the GISTs showed severe hyalinization with calcification and partial heterotopic ossification, which may have caused the patient's severe dysphagia. Mutational analysis of the c-kit gene revealed a substitution at codon 642 in exon 13 (K642T) in the tumor, normal ileal mucosa and peripheral blood leukocytes, indicating that the mutation is in the germline. This is the first case of multiple GISTs with novel germline c-kit gene mutation at exon 13.

Gastric cancer is one of the most common malignancies worldwide and patients with advanced gastric cancer still have poor clinical outcomes. The overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) mRNA in colorectal cancer and its correlation with clinicopathological factors were recently reported by us. In this study, we show LGR5 mRNA overexpression in human gastric cancer specimens by quantitative RT-PCR and in situ hybridization and assess a correlation with clinicopathological factors. The mean expression of LGR5 mRNA in cancerous tissues was five times higher than that in normal tissue (P = 0.0002). Furthermore, LGR5 mRNA expression show marked variation among cases and significantly increased in cases where lymphatic invasion was present compared with those where it was absent (P = 0.0056). Although the mean expression level of LGR5 was observed to be higher in nodal metastasis and venous invasion positive cases compared to negative cases, a significant difference was not observed. These results suggest that LGR5 can be a biomarker for malignancy in gastric cancer.

Few cases of gastric carcinoma with infiltrating multi-nucleated giant cells (MGCs) have been reported, and giant cells infiltrating the gastric carcinoma were previously described as osteoclast-like giant cells (OGCs). However, hepatoid adenocarcinomas have never been reported previously, and the present case is extremely rare. A 100-year-old Japanese man with gastralgia was found to have a mass in his gastric body. Histological examination showed a poorly differentiated adenocarcinoma with infiltration of MGCs. Vascular and lymphatic invasion were noted but there were no metastases. Almost all the tumor comprised cells with hepatoid-like features. The MGCs proliferated, with infiltration of lymphoid cells. A few MGCs contained mucous material in their cytoplasm, indicating these were foreign-body giant cells. Immunohistochemically, the hepatoid-like components were positive for AFP, and staining with polyclonal antibodies against carcinoembryonic antigen (CEA) showed the canalicular pattern. We concluded that this component was hepatoid adenocarcinoma. The MGCs were positive for CD68, and surrounding infiltrating lymphoid cells were diffusely positive for CD3. Infiltration of MGCs in gastric cancer may represent a cellular immune response against gastric cancer invasion. Further studies are required to elucidate the etiology of MGCs in gastric cancer.

Aim: The aim of this study was to clarify the significance of DNA methylation alterations during gastric carcinogenesis. Background: DNA methylation alterations are induced by carcinogenetic factors at the precancerous stage in various organs. As for gastric mucosae, aberrant DNA methylation is considered to be induced by Helicobacter pylori, Epstein-Barr virus and other carcinogenetic factors. Since field cancerization concept has become evident in the stomach, non-cancerous gastric mucosae obtained from patients with gastric carcinomas (GCs) can be at the precancerous stage. However, correlations between DNA methylation profiles in such non-cancerous gastric mucosae at the precancerous stage and clinicopathological aggressiveness of GCs developing in the individual same patients and patient outcome have not been clarified. Methods: Single-CpG resolution genome-wide DNA methylation analysis using the Infinium HumanMethylation27 Bead Array (Illumina) was performed in 110 paired samples of non-cancerous gastric mucosa (N) and tumorous tissue (T) from patients with GCs. Results: DNA methylation alterations on 3,877 probes occurred in T samples relative to N samples. Since Ns can be at the precancerous stage according to field cancerization concept, we first focused on DNA methylation levels in N samples (βN) and performed unsupervised hierarchical clustering using βN on the 3,877 probes. 110 patients with GCs were subclustered into Cluster A (n=20), Cluster B1 (n=20) and Cluster B2 (n=70). GCs belonging to Cluster B1 more frequently showed undifferentiated histology and higher pT stage and pathological TNM stage when compared to those in Clusters A and B2. Recurrence-free and overall survival rates of patients in Cluster B1 were lower than those of patients in Clusters A and B2. Sixty hallmark genes of which βN characterized the epigenetic clustering were identified. To examine whether DNA methylation profiles in N samples were inherited by GCs themselves, we next focused on DNA methylation levels of the 60 hallmark genes in T samples (βT). In 43 out of the 60 hallmark genes, βT was again significantly correlated with undifferentiated histology or higher pT stage and pathological TNM stage. In 25 and 26 out of the 60 hallmark genes, βT was significantly correlated with recurrence-free and overall survival rates of the 110 patients, respectively. Multivariate analyses using Cox proportional hazards regression model revealed that βT in 10 out of the 60 hallmark genes were a significant prognostic factor which was independent from histological differentiation, pT stage and pathological TNM stage. Conclusion: These data indicated that DNA methylation profiles at the precancerous stage may be inherited by GCs themselves and may determine tumor aggressiveness and patient outcome. Citation Format: Kazuhiro Yamanoi, Eri Arai, Yoriko Takahashi, Sayaka Miyata, Ryoji Kushima, Hitoshi Katai, Michiie Sakamoto, Yae Kanai. Epigenetic clustering of gastric carcinoma based on DNA methylation profiles at the precancerous stage: its correlation with tumor aggressiveness and patient outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1370. doi:10.1158/1538-7445.AM2014-1370